Ohmori Shiho, Kawai Atsuko, Takada Nobuhiro, Saito-Ito Atsuko
Department of Pharmacy, School of Pharmacy, Hyogo University of Health Sciences, Kobe, Hyogo, Japan.
Parasitol Int. 2011 Dec;60(4):403-9. doi: 10.1016/j.parint.2011.06.021. Epub 2011 Jun 25.
We have developed a real-time PCR assay that can rapidly and differentially detect and quantify four genotypes of small subunit ribosomal RNA gene (SSUrDNA) of Babesia microti (Kobe-, Otsu-, Nagano- and US-types). In this assay, four genotype-specific pairs of primers targeted on internal transcribed spacer (ITS) 1 or 2 sequences were used and amplicons by each pair of primers were quantitatively detected by fluorescent SYBR Green I. The four genotype-specific pairs of primers displayed the high specificity for homologous genotype DNA. The standard curves of cycle threshold (Ct) values versus amount of target DNA per reaction (log) for all four genotypes were linear and the correlation coefficient (Rsq) values for the curves were from 0.970 to 0.997. The standard curves were almost identical even in the presence of heterologous genotype DNA. This assay could detect 10-30 fg purified DNA (equivalent to the amount of 1-5 parasite DNA) of each genotype B. microti. This assay could also detect each genotype B. microti infection in blood with 3×10(-6)%-1×10(-5)% parasitemia. This assay was applicable to field rodent and tick samples to reveal mixed infection in several samples, for which a single genotype of B. microti had been detected by direct sequencing analyses in our previous studies. This assay also seemed to be applicable to clinical human samples, showing Kobe-type positive results for the first Japanese babesiosis patient and the asymptomatic donor, both infected with Kobe-type B. microti.
我们开发了一种实时聚合酶链反应（PCR）检测方法，该方法能够快速、区分检测并定量微小巴贝斯虫（Babesia microti）小亚基核糖体RNA基因（SSUrDNA）的四种基因型（神户型、大津型、长野型和美国型）。在该检测方法中，使用了针对内转录间隔区（ITS）1或2序列的四对基因型特异性引物，并且通过荧光SYBR Green I对每对引物的扩增子进行定量检测。这四对基因型特异性引物对同源基因型DNA表现出高度特异性。所有四种基因型的循环阈值（Ct）值与每个反应中目标DNA量（对数）的标准曲线均呈线性，曲线的相关系数（Rsq）值在0.970至0.997之间。即使存在异源基因型DNA，标准曲线也几乎相同。该检测方法能够检测出每种微小巴贝斯虫基因型10-30 fg的纯化DNA（相当于1-5个寄生虫DNA的量）。该检测方法还能够检测出血液中微小巴贝斯虫每种基因型感染，其寄生虫血症为3×10（-6）%-1×10（-5）%。该检测方法适用于野外啮齿动物和蜱虫样本，以揭示多个样本中的混合感染情况，在我们之前的研究中，通过直接测序分析已在这些样本中检测到单一基因型的微小巴贝斯虫。该检测方法似乎也适用于临床人类样本，对首例感染神户型微小巴贝斯虫的日本巴贝斯虫病患者和无症状捐赠者均显示出神户型阳性结果。
